CN104950060B - Based on chromatograph-spectrogrph combination and the analysis method of the paeonol content of subspace angle criterion - Google Patents
Based on chromatograph-spectrogrph combination and the analysis method of the paeonol content of subspace angle criterion Download PDFInfo
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- UILPJVPSNHJFIK-UHFFFAOYSA-N Paeonol Chemical compound COC1=CC=C(C(C)=O)C(O)=C1 UILPJVPSNHJFIK-UHFFFAOYSA-N 0.000 title claims abstract description 250
- YLTGFGDODHXMFB-UHFFFAOYSA-N isoacetovanillon Natural products COC1=CC=C(C(C)=O)C=C1O YLTGFGDODHXMFB-UHFFFAOYSA-N 0.000 title claims abstract description 125
- MLIBGOFSXXWRIY-UHFFFAOYSA-N paeonol Natural products COC1=CC=C(O)C(C(C)=O)=C1 MLIBGOFSXXWRIY-UHFFFAOYSA-N 0.000 title claims abstract description 125
- 238000004458 analytical method Methods 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 22
- 238000001228 spectrum Methods 0.000 claims abstract description 17
- 238000002211 ultraviolet spectrum Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 230000003595 spectral effect Effects 0.000 claims description 61
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 37
- 230000010354 integration Effects 0.000 claims description 15
- 239000011159 matrix material Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000012452 mother liquor Substances 0.000 claims description 11
- 238000002425 crystallisation Methods 0.000 claims description 10
- 230000008025 crystallization Effects 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 claims description 8
- 238000004364 calculation method Methods 0.000 claims description 8
- 238000004811 liquid chromatography Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 5
- 239000013558 reference substance Substances 0.000 claims description 5
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- 238000000825 ultraviolet detection Methods 0.000 claims description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 2
- 230000008878 coupling Effects 0.000 claims 3
- 238000010168 coupling process Methods 0.000 claims 3
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- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 7
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- 239000000523 sample Substances 0.000 description 50
- 238000011084 recovery Methods 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 2
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- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 1
- 241000736199 Paeonia Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 240000005001 Paeonia suffruticosa Species 0.000 description 1
- 235000003889 Paeonia suffruticosa Nutrition 0.000 description 1
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Abstract
本发明公开了一种基于色谱‑光谱仪联用和子空间夹角判据的丹皮酚含量的分析方法，具体按照以下步骤实施：丹皮酚标准光谱库建立，待测样本的制备及紫外光谱采集，建模本底数据库的建立，待测样本中丹皮酚含量测定。本文通过液相‑紫外可见光谱仪联用，应用子空间‑夹角判据，建立了丹皮酚含量快速分析方法。对于同类样品的测定，而且只需一次建模便可批量检测。与高效液相色谱法相比，所建立的方法稳定可靠、操作简便，样品不需要复杂的前处理，为丹皮酚含量快速测定提供了一种比较实用的定量分析技术，并可推广应用于其它丹皮酚制品检测领域。
The invention discloses an analysis method of paeonol content based on the combination of chromatography-spectroscopy and the subspace angle criterion, which is specifically implemented according to the following steps: establishment of paeonol standard spectrum library, preparation of samples to be tested and collection of ultraviolet spectra , the establishment of the modeling background database, and the determination of paeonol content in the samples to be tested. In this paper, a rapid analysis method for paeonol content was established by combining liquid phase-ultraviolet-visible spectrometer and applying the subspace-angle criterion. For the determination of the same type of samples, it can be tested in batches with only one modeling. Compared with high performance liquid chromatography, the established method is stable, reliable, easy to operate, and the sample does not require complicated pretreatment. It provides a more practical quantitative analysis technique for the rapid determination of paeonol content, and can be extended to other Paeonol products testing field.
Description
技术领域technical field
本发明属于药物分析技术领域，具体涉及一种基于色谱-光谱仪联用和子空间夹角判据的丹皮酚含量的分析方法。The invention belongs to the technical field of drug analysis, and in particular relates to an analysis method for paeonol content based on the combined use of a chromatography-spectrometer and a subspace angle criterion.
背景技术Background technique
丹皮酚是从中国的国花牡丹花的根皮中提取物出来的一种中药材。提取自毛茛科植物牡丹的干燥根皮。丹皮酚药理应用价值广泛，除用于医疗制剂原料外，还用于牙膏、护肤、美容等日化品中。丹皮酚的含量测定是各种制剂和产品质量控制的主要指标之一。目前丹皮酚含量分析多采用高效液相色谱法，该法敏度高，但分析成本高，操作强度大，大批量样本的分析效率低，需要建立一种丹皮酚含量快速分析新方法。Paeonol is a traditional Chinese medicine extracted from the root bark of the national flower of China, peony. Extracted from the dried root bark of the Ranunculaceae tree peony. Paeonol has a wide range of pharmacological applications. In addition to being used as raw materials for medical preparations, it is also used in toothpaste, skin care, beauty and other daily chemicals. The content determination of paeonol is one of the main indicators of various preparations and product quality control. At present, high performance liquid chromatography is mostly used for the analysis of paeonol content. This method has high sensitivity, but the analysis cost is high, the operation intensity is high, and the analysis efficiency of large batches of samples is low. It is necessary to establish a new method for rapid analysis of paeonol content.
发明内容Contents of the invention
本发明的目的是提供一种基于色谱-光谱仪联用和子空间夹角判据的丹皮酚含量的分析方法，解决了现有技术中存在的分析效率低、操作强度大以及分析成本高的问题。The purpose of the present invention is to provide a method for analyzing paeonol content based on the combination of chromatography-spectroscopy and the subspace angle criterion, which solves the problems of low analysis efficiency, high operation intensity and high analysis cost in the prior art .
本发明所采用的技术方案是，一种基于色谱-光谱仪联用和子空间夹角判据的丹皮酚含量的分析方法，具体按照以下步骤实施：The technical scheme adopted in the present invention is, a kind of analysis method of the content of paeonol based on chromatograph-spectrometer combination and subspace angle criterion, specifically implement according to the following steps:
步骤1、丹皮酚标准光谱库建立，Step 1, paeonol standard spectral library is established,
步骤2、待测样本的制备及紫外光谱采集，Step 2, the preparation of the sample to be tested and the collection of ultraviolet spectrum,
步骤3、建模本底数据库的建立，Step 3, the establishment of the modeling background database,
步骤4、待测样本中丹皮酚含量测定。Step 4, paeonol content determination in the sample to be tested.
本发明的特点还在于，The present invention is also characterized in that,
丹皮酚标准光谱库建立具体为：配制系列浓度为0.2～20mg/L的丹皮酚标准溶液，以75％乙醇为空白，采集系列溶液的190nm-1100nm波长范围光谱；选择200nm-400nm波长范围光谱，经多变量最小二乘回归后得到丹皮酚标准光谱yi＝aix+bi，记为V，其中在270nm处的线性方程和相关系数分别为：y＝0.0792x-0.0636；r＝0.9992。The establishment of the paeonol standard spectral library is as follows: prepare a series of paeonol standard solutions with a concentration of 0.2-20 mg/L, use 75% ethanol as a blank, and collect the spectra of the 190nm-1100nm wavelength range of the series of solutions; select the 200nm-400nm wavelength range Spectrum, paeonol standard spectrum y i =a i x+ bi obtained after multivariate least squares regression, denoted as V, wherein the linear equation and correlation coefficient at 270nm are respectively: y=0.0792x-0.0636; r=0.9992.
待测样本的制备及紫外光谱采集具体为：取丹皮酚结晶母液，室温下，分别取0.15g上层溶液和下层晶体各一份，用75％乙醇稀释到浓度为10-20mg/L，得到样本S1和S2；The preparation of the sample to be tested and the collection of ultraviolet spectrum are as follows: take the mother liquor of paeonol crystallization, at room temperature, take 0.15g of the upper layer solution and a portion of the lower layer crystal respectively, dilute with 75% ethanol to a concentration of 10-20mg/L, and obtain samples S1 and S2 ;
取不同生产批次的丹皮酚结晶母液两份，分别加热至50℃成熔融状态，分别称取1.2g和0.9g，用75％乙醇稀释到浓度为10-20mg/L，得到样本S3和S4；Take two parts of paeonol crystallization mother liquor from different production batches, heat them to 50°C respectively to melt, weigh 1.2g and 0.9g respectively, and dilute with 75% ethanol to a concentration of 10-20mg/L to obtain sample S 3 and S4 ;
称取丹皮酚结晶产品0.1g，用75％乙醇稀释到浓度为10-20mg/L，得到样本S5；取S1-S5等体积混合得到样本S6；Weigh 0.1 g of the paeonol crystalline product and dilute it with 75% ethanol to a concentration of 10-20 mg/L to obtain a sample S 5 ; mix equal volumes of S 1 -S 5 to obtain a sample S 6 ;
采集S1-S6的紫外光谱数据，得到待测样本数据库，记为D；积分时间15微秒，积分次数20次，波长200-400nm。Collect the ultraviolet spectrum data of S 1 -S 6 to obtain the sample database to be tested, denoted as D; the integration time is 15 microseconds, the number of integration times is 20, and the wavelength is 200-400nm.
步骤3建模本底数据库的建立具体为：对待测样品S1-S6和丹皮酚对照品样本B3，通过液相色谱与光谱仪联用，采集各样本经过色谱柱完全分离后的多波长光谱数据；从待测样品光谱数据中扣除待测组分丹皮酚的光谱数据，并经数据降维，得到本底光谱数据库N；The establishment of the modeling background database in step 3 is as follows: for the samples S 1 -S 6 to be tested and the paeonol reference substance sample B 3 , use liquid chromatography and spectrometer to collect multiple samples after they are completely separated by the chromatographic column. Wavelength spectral data; subtract the spectral data of the component paeonol to be measured from the spectral data of the sample to be measured, and reduce the dimension of the data to obtain the background spectral database N;
液相色谱条件:4.6mm×150mm C18柱；梯度洗脱，流动相：0～30min，乙腈：水＝40：60(V/V)；30～55min，乙腈：水＝5：95(V/V)；55～80min，乙腈：水＝95：5(V/V)；流速1mL/min；进样量20μL；柱温20℃；色谱检测波长270nm；光谱条件：流动比色皿1cm；积分时间：15微秒；积分次数：20次；紫外检测波长：200-400nm。Liquid chromatography conditions: 4.6mm × 150mm C 18 column; gradient elution, mobile phase: 0 ~ 30min, acetonitrile: water = 40: 60 (V / V); 30 ~ 55min, acetonitrile: water = 5: 95 (V /V); 55～80min, acetonitrile:water=95:5(V/V); flow rate 1mL/min; injection volume 20μL; column temperature 20℃; chromatographic detection wavelength 270nm; Integration time: 15 microseconds; integration times: 20 times; UV detection wavelength: 200-400nm.
本底光谱数据库的降维中，所述本底光谱数据库W降低到适当的维数的方法如下：应用[U，S，V]＝svd(W)对本底光谱数据库W进行奇异值分解降维，分解后得到m阶行正交矩阵U、n阶列正交矩阵V和奇异值矩阵S，取U的前q列，为降维后的本底数据库N。In the dimensionality reduction of the background spectral database, the method for reducing the background spectral database W to an appropriate dimension is as follows: apply [U, S, V]=svd(W) to carry out singular value decomposition and dimensionality reduction to the background spectral database W , after decomposing, the m-order row-orthogonal matrix U, the n-order column-orthogonal matrix V and the singular value matrix S are obtained. The first q columns of U are taken as the background database N after dimensionality reduction.
待测样本中丹皮酚含量测定具体为：将上述标准光谱数据库V，本底光谱数据库N以及D导入计算平台，选取200-400nm波长段，应用向量-子空间夹角判据算法分别计算各待测样本中丹皮酚的含量。The determination of the content of paeonol in the sample to be tested is specifically as follows: import the above-mentioned standard spectral database V, background spectral database N and D into the computing platform, select the 200-400nm wavelength range, and apply the vector-subspace included angle criterion algorithm to calculate each The content of paeonol in the sample to be tested.
待测样本中丹皮酚含量测定的具体步骤为：The concrete steps of paeonol content determination in the sample to be tested are:
步骤4.1、依据定量精度设定扣减步长Δ；Step 4.1. Set the deduction step size Δ according to the quantitative accuracy;
步骤4.2、在算式yi＝aix+bi中带入较大的x1值，得到v1；Step 4.2, put a larger value of x 1 into the formula y i =a i x+ bi to obtain v 1 ;
所述的yi表示在i波长下丹皮酚的吸光度值，ai、bi是常数，x表示对羟基苯甲酸的浓度，v1表示在浓度为x1时丹皮酚的多波长吸光度值y1，v1为所有的yi值组成的矩阵；Described y i represents the absorbance value of paeonol at i wavelength, a i and b i are constants, x represents the concentration of p-hydroxybenzoic acid, and v1 represents the multi-wavelength absorbance of paeonol when the concentration is x1 Value y 1 , v 1 is a matrix composed of all y i values;
步骤4.3、从待测样本光谱数据a中扣除v1/Δ，扣除后的变量记为da；把本底光谱数据库N和变量da合并后记为对比空间M，计算对比空间M与v1夹角；Step 4.3. Deduct v 1 /Δ from the spectral data a of the sample to be tested, and record the variable after deduction as da; combine the background spectral database N and the variable da and record it as the comparison space M, and calculate the angle between the comparison space M and v 1 ;
步骤4.4、从待测样本光谱数据a中逐步扣除v1后，重复步骤(3)；Step 4.4, after gradually deducting v1 from the spectral data a of the sample to be tested, repeat step (3);
步骤4.5、当待测样本中的丹皮酚完全被扣除后，比对空间M和丹皮酚的光谱向量v1的空间夹角值会出现最大值θmax，记录空间夹角最大值θmax出现时对应的扣减步数λ，通过丹皮酚的浓度x1和扣减步数估算待测样本中丹皮酚的含量Y1，计算式为Y1＝X1×λ/Δ，得到的Y1即为待测样本中丹皮酚的含量值。Step 4.5, when the paeonol in the sample to be tested is completely deducted, the maximum value of the space angle between the comparison space M and the spectral vector v 1 of paeonol will appear θ max , and the maximum value of the space angle θ max will be recorded The corresponding number of deduction steps λ when it appears, the paeonol content Y 1 in the sample to be tested is estimated by the concentration of paeonol x 1 and the number of deduction steps, and the calculation formula is Y 1 =X 1 ×λ/Δ, which can be obtained Y1 is the content value of paeonol in the sample to be tested.
本发明的有益效果是：本文首先构建丹皮酚标准数据库，然后通过对不同批次丹皮酚结晶母液样品进行一次高效液相色谱-光谱仪联用，采集各样品经色谱分离后的光谱数据，经数据处理获得不含被测组分丹皮酚的本底数据，结合空间夹角判据算法可实现直接分析待测样本中丹皮酚含量。本文通过液相-紫外可见光谱仪联用，应用子空间-夹角判据，建立了丹皮酚含量快速分析方法。对于同类样品的测定，而且只需一次建模便可批量检测。与高效液相色谱法相比，所建立的方法稳定可靠、操作简便，样品不需要复杂的前处理，为丹皮酚含量快速测定提供了一种比较实用的定量分析技术，并可推广应用于其它丹皮酚制品检测领域。The beneficial effects of the present invention are: firstly, the paeonol standard database is constructed in this paper, and then the spectral data of each sample after chromatographic separation are collected by performing a high performance liquid chromatography-spectroscopy on different batches of paeonol crystallization mother liquor samples, After data processing, the background data without paeonol, the measured component, was obtained, and combined with the spatial angle criterion algorithm, the content of paeonol in the sample to be tested could be directly analyzed. In this paper, a rapid analysis method for paeonol content was established through the combination of liquid phase-ultraviolet-visible spectrometer and the subspace-angle criterion. For the determination of the same type of samples, it can be tested in batches with only one modeling. Compared with high performance liquid chromatography, the established method is stable, reliable, easy to operate, and the sample does not require complicated pretreatment. It provides a more practical quantitative analysis technique for the rapid determination of paeonol content, and can be extended to other Paeonol products testing field.
附图说明Description of drawings
图1是丹皮酚母液和丹皮酚对照品的液相色谱图。Fig. 1 is the liquid chromatogram of paeonol mother liquor and paeonol reference substance.
具体实施方式detailed description
下面结合具体实施方式对本发明进行详细说明。The present invention will be described in detail below in combination with specific embodiments.
本发明提供一种基于色谱-光谱仪联用和子空间夹角判据的丹皮酚含量的分析方法，具体按照以下步骤实施：The present invention provides a method for analyzing the content of paeonol based on the combination of chromatography-spectroscopy and the subspace angle criterion, which is specifically implemented according to the following steps:
步骤1、丹皮酚标准光谱库建立，Step 1, paeonol standard spectral library is established,
配制系列浓度为0.2～20mg/L的丹皮酚标准溶液，以75％乙醇为空白，采集系列溶液的190nm-1100nm波长范围光谱；选择200nm-400nm波长范围光谱，经多变量最小二乘回归后得到丹皮酚标准光谱yi＝aix+bi，记为V，其中在270nm处的线性方程和相关系数分别为：y＝0.0792x-0.0636；r＝0.9992。Prepare a series of paeonol standard solutions with a concentration of 0.2 to 20 mg/L, use 75% ethanol as a blank, and collect the 190nm-1100nm wavelength spectrum of the series of solutions; select the 200nm-400nm wavelength range spectrum, after multivariate least squares regression The paeonol standard spectrum y i =a i x+ bi is obtained, denoted as V, where the linear equation and correlation coefficient at 270nm are: y=0.0792x-0.0636; r=0.9992.
步骤2、待测样本的制备及紫外光谱采集，Step 2, the preparation of the sample to be tested and the collection of ultraviolet spectrum,
取丹皮酚结晶母液，室温下，分别取0.15g左右上层溶液和下层晶体各一份，用75％乙醇稀释到浓度为10-20mg/L，得到样本S1和S2。Take the mother liquor of paeonol crystallization, take about 0.15g of the upper layer solution and one portion of the lower layer crystal at room temperature, and dilute with 75% ethanol to a concentration of 10-20mg/L to obtain samples S 1 and S 2 .
取不同生产批次的丹皮酚结晶母液两份，分别加热至50℃成熔融状态，分别称取1.2g和0.9g，用75％乙醇稀释到10-20mg/L，得到样本S3和S4。Take two parts of paeonol crystallization mother liquor from different production batches, heat them to 50°C to melt, weigh 1.2g and 0.9g respectively, and dilute to 10-20mg/L with 75% ethanol to obtain samples S 3 and S 4 .
称取丹皮酚结晶产品0.1g，用75％乙醇稀释到10-20mg/L，得到样本S5；取S1-S5等体积混合得到样本S6。Weigh 0.1 g of paeonol crystalline product and dilute it to 10-20 mg/L with 75% ethanol to obtain sample S 5 ; take equal volumes of S 1 -S 5 and mix them to obtain sample S 6 .
采集S1-S6的紫外光谱数据，得到待测样本数据库，记为D。积分时间15微秒；积分次数20次；波长200-400nm。Collect the ultraviolet spectrum data of S 1 -S 6 to obtain the sample database to be tested, denoted as D. The integration time is 15 microseconds; the number of integrations is 20 times; the wavelength is 200-400nm.
步骤3、建模本底数据库的建立，Step 3, the establishment of the modeling background database,
对待测样品S1-S6和丹皮酚对照品样本B3，通过液相色谱与光谱仪联用，采集各样本经过色谱柱完全分离后的多波长光谱数据，从待测样品光谱数据中扣除待测组分丹皮酚的光谱数据，并经数据降维，得到本底光谱数据库N。For the samples S 1 -S 6 to be tested and the paeonol reference sample B 3 , use liquid chromatography and spectrometer to collect the multi-wavelength spectral data of each sample after being completely separated by the chromatographic column, and deduct them from the spectral data of the sample to be tested. The spectral data of paeonol, the component to be measured, was obtained through data dimensionality reduction to obtain the background spectral database N.
液相色谱条件:C18柱(4.6mm×150mm)；梯度洗脱，流动相：0～30min，乙腈：水＝40：60(V/V)；30～55min，乙腈：水＝5：95(V/V)；55～80min，乙腈：水＝95：5(V/V)。流速1mL/min；进样量20μL；柱温20℃；色谱检测波长270nm。Liquid chromatography conditions: C 18 column (4.6mm×150mm); gradient elution, mobile phase: 0-30min, acetonitrile: water = 40:60 (V/V); 30-55min, acetonitrile: water = 5:95 (V/V); 55-80min, acetonitrile: water = 95:5 (V/V). The flow rate is 1mL/min; the injection volume is 20μL; the column temperature is 20°C; the chromatographic detection wavelength is 270nm.
光谱条件：流动比色皿(1cm)；积分时间：15微秒；积分次数：20次；紫外检测波长：200-400nm。Spectral conditions: mobile cuvette (1cm); integration time: 15 microseconds; integration times: 20 times; UV detection wavelength: 200-400nm.
步骤4、待测样本中丹皮酚含量测定。Step 4, paeonol content determination in the sample to be tested.
将上述标准光谱数据库V，本底光谱数据库N以及D导入Matlab计算平台，选取200-400nm波长段，应用向量-子空间夹角判据算法(简称VS法)分别计算各待测样本中丹皮酚的含量；Import the above-mentioned standard spectral database V, background spectral database N and D into the Matlab computing platform, select the 200-400nm wavelength segment, and apply the vector-subspace angle criterion algorithm (abbreviated as VS method) to calculate the paeonol in each sample to be tested. phenolic content;
其中，待测样本中丹皮酚含量测定具体为：Wherein, the determination of paeonol content in the sample to be tested is specifically:
步骤4.1、依据定量精度设定扣减步长Δ；Step 4.1. Set the deduction step size Δ according to the quantitative accuracy;
步骤4.2、在算式yi＝aix+bi中带入较大的x1值，得到v1；Step 4.2, put a larger value of x 1 into the formula y i =a i x+ bi to obtain v 1 ;
所述的yi表示在i波长下丹皮酚的吸光度值，ai、bi是常数，x表示丹皮酚的浓度，v1表示在浓度为x1时丹皮酚的多波长吸光度值y1，v1为所有的yi值组成的矩阵；The y i represent the absorbance value of paeonol at i wavelength, a i and b i are constants, x represents the concentration of paeonol, and v1 represents the multi-wavelength absorbance value of paeonol when the concentration is x1 y 1 , v 1 is a matrix composed of all y i values;
步骤4.3、从待测样本光谱数据a中扣除v1/Δ，扣除后的变量记为da；把本底光谱数据库N和变量da合并后记为对比空间M，计算对比空间M与v1夹角；Step 4.3. Deduct v 1 /Δ from the spectral data a of the sample to be tested, and record the variable after deduction as da; combine the background spectral database N and the variable da and record it as the comparison space M, and calculate the angle between the comparison space M and v 1 ;
步骤4.4、从待测样本光谱数据a中逐步扣除v1后，重复步骤4.3；Step 4.4, after gradually deducting v1 from the spectral data a of the sample to be tested, repeat step 4.3;
步骤4.5、当待测样本中的丹皮酚完全被扣除后，比对空间M和丹皮酚的光谱向量v1的空间夹角值会出现最大值θmax，记录空间夹角最大值θmax出现时对应的扣减步数λ，通过丹皮酚的浓度x1和扣减步数估算待测样本中丹皮酚的含量Y1，计算式为Y1＝X1×λ/Δ，得到的Y1即为待测样本中丹皮酚的含量值。Step 4.5, when the paeonol in the sample to be tested is completely deducted, the maximum value of the space angle between the comparison space M and the spectral vector v 1 of paeonol will appear θ max , and the maximum value of the space angle θ max will be recorded The corresponding number of deduction steps λ when it appears, the paeonol content Y 1 in the sample to be tested is estimated by the concentration of paeonol x 1 and the number of deduction steps, and the calculation formula is Y 1 =X 1 ×λ/Δ, which can be obtained Y1 is the content value of paeonol in the sample to be tested.
实施例1Example 1
1实验部分1 Experimental part
1.1仪器与试剂1.1 Instruments and reagents
高效液相色谱仪(LC-20AT，岛津)；紫外-可见光纤光谱仪(Maya2000，Ocean Optics)；电子分析天平(AL104，梅特勒-托利多)；超声波清洗机(上海之信仪器有限公司)；无水乙醇(AR)，甲醇(色谱纯)，乙腈(色谱纯)，丹皮酚对照品(纯度99.9％)，丹皮酚结晶母液(广西亿康制药业有限公司)。High-performance liquid chromatography (LC-20AT, Shimadzu); UV-visible fiber optic spectrometer (Maya2000, Ocean Optics); electronic analytical balance (AL104, Mettler-Toledo); ultrasonic cleaning machine (Shanghai Zhixin Instrument Co., Ltd. ); absolute ethanol (AR), methanol (chromatographically pure), acetonitrile (chromatographically pure), paeonol reference substance (purity 99.9%), paeonol crystallization mother liquor (Guangxi Yikang Pharmaceutical Industry Co., Ltd.).
1.2试验方法1.2 Test method
1.2.1丹皮酚标准光谱库建立1.2.1 Establishment of paeonol standard spectral library
精确称取0.1010g丹皮酚对照品，用75％乙醇定容到100mL。依次配制浓度为2mg/L、4mg/L、8mg/L、12mg/L、16mg/L、20mg/L丹皮酚-75％乙醇溶液B1-B6。采集各溶液200nm-400nm紫外光谱，经最小二乘回归得到标准光谱库V。Accurately weigh 0.1010g paeonol reference substance, and dilute to 100mL with 75% ethanol. Concentrations of 2mg/L, 4mg/L, 8mg/L, 12mg/L, 16mg/L, 20mg/L paeonol-75% ethanol solutions B 1 -B 6 were sequentially prepared. The 200nm-400nm ultraviolet spectrum of each solution was collected, and the standard spectral library V was obtained by least squares regression.
1.2.2待测样本的制备及紫外光谱采集1.2.2 Preparation of samples to be tested and collection of ultraviolet spectra
取生产企业提供的丹皮酚结晶母液，室温下，分别取0.15g左右上层溶液和下层晶体各一份，用75％乙醇稀释到一定浓度，得到样本S1和S2。Take the mother liquor of paeonol crystallization provided by the manufacturer, take about 0.15g of the upper layer solution and one portion of the lower layer crystal at room temperature, and dilute to a certain concentration with 75% ethanol to obtain samples S 1 and S 2 .
取不同生产批次的丹皮酚结晶母液两份，分别加热至50℃成熔融状态，分别称取1.2g和0.9g，用75％乙醇稀释到一定浓度，得到样本S3和S4。Two parts of paeonol crystallization mother liquor from different production batches were taken, heated to 50°C to melt, 1.2g and 0.9g were weighed, and diluted to a certain concentration with 75% ethanol to obtain samples S 3 and S 4 .
称取丹皮酚结晶产品0.1g，用75％乙醇稀释到一定浓度，得到样本S5。取S1-S5等体积混合得到样本S6。Weigh 0.1 g of paeonol crystalline product and dilute to a certain concentration with 75% ethanol to obtain sample S 5 . Equal volumes of S 1 -S 5 were mixed to obtain sample S 6 .
采集S1-S6的紫外光谱数据，得到待测样本数据库，记为D。积分时间15微秒；积分次数20次；波长200-400nm。Collect the ultraviolet spectrum data of S 1 -S 6 to obtain the sample database to be tested, denoted as D. The integration time is 15 microseconds; the number of integrations is 20 times; the wavelength is 200-400nm.
1.2.3建模本底数据库的建立1.2.3 Establishment of modeling background database
对待测样品S1-S6和丹皮酚对照品样本B3，通过液相色谱与光谱仪联用，采集各样本经过色谱柱完全分离后的多波长光谱数据。从待测样品光谱数据中扣除待测组分丹皮酚的光谱数据，并经数据降维，得到本底光谱数据库N。For the samples S 1 -S 6 to be tested and the paeonol reference sample B 3 , the multi-wavelength spectral data of each sample after being completely separated by the chromatographic column was collected through liquid chromatography and a spectrometer. The spectral data of paeonol, the component to be tested, is subtracted from the spectral data of the sample to be tested, and the background spectral database N is obtained through data dimensionality reduction.
液相色谱条件:C18柱(4.6mm×150mm)；梯度洗脱，流动相：0～30min，乙腈：水＝40：60(V/V)；30～55min，乙腈：水＝5：95(V/V)；55～80min，乙腈：水＝95：5(V/V)。流速1mL/min；进样量20μL；柱温20℃；色谱检测波长270nm。Liquid chromatography conditions: C 18 column (4.6mm×150mm); gradient elution, mobile phase: 0-30min, acetonitrile: water = 40:60 (V/V); 30-55min, acetonitrile: water = 5:95 (V/V); 55-80min, acetonitrile: water = 95:5 (V/V). The flow rate is 1mL/min; the injection volume is 20μL; the column temperature is 20°C; the chromatographic detection wavelength is 270nm.
光谱条件：流动比色皿(1cm)；积分时间：15微秒；积分次数：20次；紫外检测波长：200-400nm。Spectral conditions: mobile cuvette (1cm); integration time: 15 microseconds; integration times: 20 times; UV detection wavelength: 200-400nm.
1.2.4待测样本中丹皮酚含量测定1.2.4 Determination of paeonol content in the sample to be tested
将上述标准光谱数据库V，本底光谱数据库N以及D导入计算平台，选取200-400nm波长段，应用向量-子空间夹角判据算法(简称VS法)分别计算各待测样本中丹皮酚的含量。其中，待测样本中丹皮酚含量测定具体为：Import the above-mentioned standard spectral database V, background spectral database N and D into the computing platform, select the 200-400nm wavelength segment, and apply the vector-subspace angle criterion algorithm (VS method for short) to calculate the paeonol in each sample to be tested. content. Wherein, the determination of paeonol content in the sample to be tested is specifically:
步骤4.1、依据定量精度设定扣减步长Δ(本实施例为1000)；Step 4.1, set the deduction step size Δ (1000 in this embodiment) according to the quantitative accuracy;
步骤4.2、在算式yi＝aix+bi中带入较大的x1值，得到v1；Step 4.2, put a larger value of x 1 into the formula y i =a i x+ bi to obtain v 1 ;
所述的yi表示在i波长下丹皮酚的吸光度值，ai、bi是常数，x表示丹皮酚的浓度，v1表示在浓度为x1时丹皮酚的多波长吸光度值y1，v1为所有的yi值组成的矩阵；The y i represent the absorbance value of paeonol at i wavelength, a i and b i are constants, x represents the concentration of paeonol, and v1 represents the multi-wavelength absorbance value of paeonol when the concentration is x1 y 1 , v 1 is a matrix composed of all y i values;
步骤4.3、从待测样本光谱数据a中扣除v1/Δ(本实施例是扣除Δv1＝v1/1000)，扣除后的变量记为da；把本底光谱数据库N和变量da合并后记为对比空间M，计算对比空间M与v1夹角；Step 4.3. Deduct v 1 /Δ from the spectral data a of the sample to be tested (in this embodiment, Δv 1 =v 1 /1000 is deducted), and the variable after the deduction is recorded as da; after combining the background spectral database N and the variable da, write For the comparison space M, calculate the angle between the comparison space M and v 1 ;
步骤4.4、从待测样本光谱数据a中逐步扣除v1后，重复步骤4.3；Step 4.4, after gradually deducting v1 from the spectral data a of the sample to be tested, repeat step 4.3;
步骤4.5、当待测样本中的丹皮酚完全被扣除后，比对空间M和丹皮酚的光谱向量v1的空间夹角值会出现最大值θmax，记录空间夹角最大值θmax出现时对应的扣减步数λ，通过丹皮酚的浓度x1和扣减步数估算待测样本中丹皮酚的含量Y1，计算式为Y1＝X1×λ/Δ，得到的Y1即为待测样本中丹皮酚的含量值。Step 4.5, when the paeonol in the sample to be tested is completely deducted, the maximum value of the space angle between the comparison space M and the spectral vector v 1 of paeonol will appear θ max , and the maximum value of the space angle θ max will be recorded The corresponding number of deduction steps λ when it appears, the paeonol content Y 1 in the sample to be tested is estimated by the concentration of paeonol x 1 and the number of deduction steps, and the calculation formula is Y 1 =X 1 ×λ/Δ, which can be obtained Y1 is the content value of paeonol in the sample to be tested.
2.3回收率及精密度实验2.3 Recovery and precision experiments
分别取10ml S2、S4和S5各三份，分别加入8mg/L、12mg/L、16mg/L三个浓度的丹皮酚对照品溶液10mL，混合均匀后采集其多波长紫外混合光谱。采用空间夹角判据计算含量值，计算该方法的回收率和精密度。Take three parts of 10ml S2, S4 and S5 respectively, add 10mL of paeonol reference solution with three concentrations of 8mg/L, 12mg/L and 16mg/L respectively, mix well and collect its multi-wavelength ultraviolet mixed spectrum . The content value was calculated using the space angle criterion, and the recovery rate and precision of the method were calculated.
3.结果与讨论3. Results and Discussion
3.1丹皮酚标准光谱库的建立3.1 Establishment of Paeonol Standard Spectral Library
配制系列浓度为0.2～20mg/L的丹皮酚标准溶液，以75％乙醇为空白，采集系列溶液的190nm-1100nm波长范围光谱。选择200nm-400nm波长范围光谱，经多变量最小二乘回归后得到丹皮酚标准光谱yi＝aix+bi，记为V，其中在270nm处的线性方程、相关系数为：y＝0.0792x-0.0636；r＝0.9992。A series of paeonol standard solutions with a concentration of 0.2-20 mg/L were prepared, and 75% ethanol was used as a blank to collect spectra in the wavelength range of 190nm-1100nm of the series of solutions. Select the 200nm-400nm wavelength range spectrum, and obtain the paeonol standard spectrum y i =a i x+ bi after multivariate least squares regression, denoted as V, wherein the linear equation and correlation coefficient at 270nm are: y= 0.0792x-0.0636; r=0.9992.
3.2本底数据库的建立3.2 Establishment of background database
对丹皮酚对照品溶液B3和混合样本S6的液相色谱图进行分析。如图1所示，A为丹皮酚的出峰时间，对应为16.3～17.5min。从S6的光谱数据中扣除与丹皮酚具有相同保留时间的光谱数据(如图1中A峰数据)，其余数据(B峰数据)则作为测定丹皮酚含量的本底数据。依次从样品S1-S5的光谱数据中分别扣除16.3～17.5min时间段的光谱数据所对应的列后存入本底数据库中，记为M1～M5。合并M1～M6数据构成数据库M。The liquid chromatograms of paeonol reference solution B 3 and mixed sample S 6 were analyzed. As shown in Figure 1, A is the peak elution time of paeonol, which corresponds to 16.3-17.5 minutes. From the spectral data of S 6 , the spectral data having the same retention time as paeonol is deducted (such as the A peak data in Figure 1), and the remaining data (B peak data) are then used as the background data for determining the paeonol content. The columns corresponding to the spectral data of the time period of 16.3 to 17.5 minutes are respectively deducted from the spectral data of samples S 1 -S 5 in turn, and stored in the background database, which are recorded as M 1 to M 5 . The database M is formed by merging the data of M 1 to M 6 .
液相-光谱联用获取的初始本底光谱数据M数据量大，若直接采用，运算时间长，影响了方法的时效性，因此需要对获取的数据进行降维以去除数据中的噪声和冗余的维数，保留效性数据、并能够提高算法的处理速度。选用主成分分析的方法判断体系主成份为8，将M经奇异值分解，取降解后U矩阵的前8列，即为降维后的本地数据库N，具体为：应用[U，S，V]＝svd(W)对本底光谱数据库W进行奇异值分解降维，分解后得到m阶行正交矩阵U、n阶列正交矩阵V和奇异值矩阵S，取U的前q列，为降维后的本底数据库N。The initial background spectral data M obtained by liquid phase-spectroscopy has a large amount of data. If it is directly used, the calculation time will be long, which affects the timeliness of the method. Therefore, it is necessary to reduce the dimensionality of the obtained data to remove noise and redundancy in the data. The remaining dimensions retain the validity data and can improve the processing speed of the algorithm. Using the method of principal component analysis to determine the principal component of the system is 8, decompose M through singular value, and take the first 8 columns of the degraded U matrix, which is the local database N after dimensionality reduction, specifically: apply [U, S, V ]=svd(W) Singular value decomposition is performed on the background spectrum database W to reduce the dimensionality. After decomposition, the row orthogonal matrix U of order m, the column orthogonal matrix V of order n and the singular value matrix S are obtained. Taking the first q columns of U, it is Base database N after dimensionality reduction.
3.3样品中丹皮酚的测定3.3 Determination of paeonol in samples
对样品样本S1～S5的紫外混合光谱数据按空间夹角判据的算法步骤计算丹皮酚的含量。同时和高效液相色谱计算出的丹皮酚含量值进行比对。通过计算，不同待测样本中的丹皮酚的含量及误差分析如表1所示。The content of paeonol was calculated according to the algorithm steps of spatial angle criterion for the ultraviolet mixed spectral data of samples S1-S5. At the same time, it was compared with the paeonol content value calculated by high performance liquid chromatography. Through calculation, the content and error analysis of paeonol in different samples to be tested are shown in Table 1.
表1两种不同方法测定结果Table 1 Determination results of two different methods
由表1可以看出采用空间夹角判据计算结果和采用高效液相色谱计算结果相接近。相对误差小于1.80％，方法的准确度较好。It can be seen from Table 1 that the calculation results using the spatial angle criterion are close to the calculation results using HPLC. The relative error is less than 1.80%, and the accuracy of the method is good.
3.4方法回收率3.4 Method recovery
回收率结果见表2。由表2可看出样品回收率范围在97.33％-102.5％，相对误差小于5.00％，RSD值为1.5218％(n＝9)。从结果可以看出，用向量－子空间夹角判据计算出来的丹皮酚的含量与实际添加量相接近，表明该方法的准确度较好。The recovery results are shown in Table 2. It can be seen from Table 2 that the sample recovery rate ranges from 97.33% to 102.5%, the relative error is less than 5.00%, and the RSD value is 1.5218% (n=9). It can be seen from the results that the content of paeonol calculated by the vector-subspace angle criterion is close to the actual addition amount, which shows that the accuracy of the method is better.
表2回收率实验Table 2 Recovery experiment
3结论3 Conclusion
本文通过液相-紫外可见光谱仪联用，应用子空间-夹角判据，建立了丹皮酚含量快速分析方法。对于同类样品的测定，而且只需一次建模便可批量检测。与高效液相色谱法相比，所建立的方法稳定可靠、操作简便，样品不需要复杂的前处理，为丹皮酚含量快速测定提供了一种比较实用的定量分析技术，并可推广应用于其它丹皮酚制品检测领域。In this paper, a rapid analysis method for paeonol content was established through the combination of liquid phase-ultraviolet-visible spectrometer and the subspace-angle criterion. For the determination of the same type of samples, it can be tested in batches with only one modeling. Compared with high performance liquid chromatography, the established method is stable, reliable, easy to operate, and the sample does not require complicated pretreatment. It provides a more practical quantitative analysis technique for the rapid determination of paeonol content, and can be extended to other Paeonol products testing field.
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| CN112086137B (en) * | 2020-08-18 | 2022-06-10 | 山东金璋隆祥智能科技有限责任公司 | Method for quantitatively analyzing sorbose content in fermentation liquor |
| CN111879717A (en) * | 2020-09-01 | 2020-11-03 | 江西纳弗堂制药有限公司 | Method for rapidly evaluating internal components and quality of traditional Chinese medicine formula granules |
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